1. ABSTRACT Kaposi's sarcoma?associated herpesvirus (KSHV) is a large DNA tumor virus that is the causative agent of Kaposi sarcoma (KS), the most prevalent AIDS-associated malignancy. KS is an angiogenic and inflammatory tumor of endothelial cell origin. It often manifests in the oral cavity, the major site for KSHV shedding and transmission via saliva. KSHV persistently infects the spindle cells of endothelial cell origin in KS tumors. In order to persist, the virus must overcome the host antiviral response. The host cells initiate antiviral response upon recognition of virus-derived moieties, including viral DNA, through innate immune sensors. Cyclic GMP- AMP synthase (cGAS) is the major sensor responsible for the recognition of cytosolic DNA. Upon binding to DNA, cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP). This cyclic dinucleotide molecule acts as second messenger to activate the adaptor STING (stimulator of interferon genes) leading to induction of interferon (IFN) antiviral responses. Although herpesviral DNA constitutes a significant PAMP (pathogen- associated molecular pattern) within incoming virions, recurring episodes of lytic replication in KS lesions cause negligible IFN antiviral responses. This observation suggests that KSHV must have evolved an effective mechanism to subvert cGAS DNA-sensing. Emerging evidence indicates that cGAS-STING pathway is not only crucial for antiviral responses but also for antitumor immunity. Therefore, elucidating the underlying mechanisms by which KSHV subverts cGAS DNA sensing is crucial for understanding KS pathobiology. We recently found that KSHV can elicit cGAS-dependent DNA sensing responses. More importantly, we revealed that a KSHV virion protein, ORF52, subverts cytosolic DNA sensing by inhibiting cGAS enzymatic activity. This is the first viral inhibitor of cGAS to be reported and we rename it KicGAS (KSHV inhibitor of cGAS). Our long- term goal is to understand how KSHV evades the host innate antiviral responses, with an emphasis on the roles of virion-contained tegument proteins. The objective here is to define the molecular mechanisms by which KicGAS inhibits cGAS, and to determine the roles of KicGAS throughout the KSHV life cycle. Our central hypothesis is that KicGAS, as a virion-contained cGAS antagonist, plays crucial roles during KSHV primary infection and lytic replication. We will test our hypothesis by pursuing the following specific aims: 1) To elucidate the molecular mechanisms by which KicGAS inhibits cGAS DNA sensing. 2) To reveal the roles of KicGAS in KSHV lytic replication. 3) To determine the roles of cGAS and KicGAS during KSHV infection of KS- relevant cells. The work proposed here is expected to offer new insights into the molecular mechanisms by which viruses subvert cGAS DNA sensing, and to reveal the roles of KicGAS in the KSHV life cycle. Such results are expected to have an important positive impact, because the identified mechanisms can aid in the development of agonists or antagonists of the cGAS-cGAMP pathway, which can then be used for therapeutic intervention to treat or prevent KSHV-associated diseases.